Light exposure was included as stress condition potentially inducing chemical modifications of the protein, followed by aggregation [Schoeneich, 2010]. The used light source emitted a spectrum ranging from 200 to 1000 nm, thus consequences on protein stability cannot be referred to a certain wavelength or the range of UV light.
Particle formation soared after day 4 (see Figure 3-6). The total numbers of particles in the µm-range exponentially increased from approximately 3 x 103 to 2 x 105 between day 4 and day 6.
Figure 3-6 – Particle counts of light exposed huAb by light obscuration.
Cumulative particle numbers ≥ diameter per mL of unstressed sample ( ), 1 day light exposed sample ( ), 4 days light exposed sample ( ), 6 days light exposed sample ( ), 7 days light exposed sample ( ), and control sample shielded from light after 7 days ( ). Error bars represent standard deviation of at least two measurements.
Turbidity measurements confirmed these observations. The formazine nephelometric units (FNU) of the irradiated samples slightly increased until day 4. The turbidity of 3.7 FNU on day 4 exceeded the benchmark of 3.2 FNU of the PhEur, below which samples are considered to be clear [PhEur 2.2.1., 2011]. This slightly increased turbidity is not caused by particles
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≤ 10 µm, since the counts of these larger particles were not significantly increased after 4 days of light exposure. Prolonging the radiation time leads to visible precipitation, enhanced formation of particles ≥ 10 µm and ≥ 25 µm and thus an escalation of turbidity. It immediately reaches the limit of detection of the method at 1300 FNU (see Figure 3-7).
Figure 3-7 – Results of turbidity measurements of human IgG1 after light exposure.
The graph shows the formazine nephelometric units of the light exposed samples (black squares) and the shielded control samples (red triangles). The error bars represent the standard deviation of three samples per time point. Within the first 4 days of irradiation turbidity slightly increased, but later on the samples on day 6 and 7 got extremely turbid. The turbidity of the control samples slightly increased within 7 days.
The total particle counts of the shielded control samples showed an increase of the species ≥ 1.0 µm, which exceeded the particle load after 4 days of light exposure, but no enhanced formation of larger particles. Therefore, the sample appeared to be clear after 7 d incubation in the radiation chamber and resulted in a slightly increased turbidity of 2.9 FNU.
The buffer formulations stressed under the same conditions showed no significant increase in total particle numbers and turbidity over 7 days of light exposure (data not shown).
The generation of particles is therefore related to the light exposure of the protein.
Dynamic light scattering of the light exposed samples showed similar results. Until 4 days of light exposure small and medium sized subvisible species were detected. The occurring heavy precipitation hindered the dynamic light scattering measurements of the samples exposed to light for 6 or 7 days. Therefore, Figure 3-8 shows only the mean particle size distribution in the nanometer range of the samples up to four days of light exposure.
The particle size distribution determined by DLS show constantly increasing Zave and PDI values over radiation time. The native huAb sample initially revealed a monodispers
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distribution with a Zave value of 11.48 nm, and a polydispersity index (PDI) of 0.042. After four days of light exposure Zave resulted in 49.31 nm and the mean PDI was 0.208. The enhanced polydispersity of these samples is represented by the additional peaks rising between 20 and 100 nm in Figure 3-8. A slight increase was also observed measuring the turbidity of these samples. 3.7 FNU were detected after 4 days of light exposure, implying that the samples are considered not to be clear anymore [PhEur 2.2.1., 2011].
Figure 3-8 – Particle size distribution of light exposed huAb samples determined by DLS.
The scattering intensity of the irradiated IgG1 samples and their diameter in nm is plotted. Each line represents the mean out of three samples, each measured in duplicate. The black solid line represents the native human IgG1 sample, the grey solid line represents the shielded control sample after 7 days, the red dashed line represents the sample after 1 day of light exposure and the blue dashed line represents the sample after 4 days of light exposure.
Within the control sample, shielded from light, a mean Zave of 47.61 nm and a PDI of 0.186 were determined after 7 days. The control samples show the formation of new particles of diameters between 30 and 400 nm with weak scattering intensities, which contribute to the slightly increase turbidity. The original peak around 11 nm represents still the main compound in this sample, maintaining more than 27 % of scattering intensity.
Besides particle formation of sub-visible and visible size range, smaller protein species like fragments or aggregates might be induced by light exposure too. Size exclusion chromatography is the most important method to detect soluble protein species and separate them concerning to their size. The results of UV detection after SEC separation of the light exposed samples of the human IgG1 antibody are summarized in Figure 3-9. The total recovery of all detected species dramatically starts to decrease on day 6 of radiation, and ends at approximately 32 % at day 7. This reduction is accompanied by the enhanced precipitation mentioned above. The recovery of monomeric species starts to decrease already on day 1, accompanied by an enhanced formation of fragments and small aggregates. On day 4, before strong formation of insoluble particles begins, 61 % of the total recovered species is composed
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of monomeric protein species, followed by 19.7 % of intermediate molecular weight species.
The relative majority of soluble high molecular weight aggregates appeared on day 6. Further radiation of the samples resulted in only half of the total recovery. Decreases in the relative amounts of all three differentiated soluble aggregate species accompanied by a constant amount of monomeric species and a slightly increasing relative amount of fragments were detected. The control samples shielded from light, maintained 97 % of total recovery over the whole study and no generation of soluble aggregates or fragments. Only an increase in subvisible particles in the size range > 1 µm < 10 µm was detected. Anyway, the tremendous formation of fragments as well as aggregates is attributed to the light exposure and not to the side effect of a slightly elevated temperature during incubation. The resulting distribution of fragments, monomeric species and the various aggregates is time dependent. Generally, with prolonged duration of irradiation, the amount and size of aggregates increases. A growth of aggregated species is suggested which sometime between day 4 and day 6 exceeds a critical size of several µm and subsequently start to precipitate. Besides aggregation, fragmentation was detected too, indicating that light exposure is also able to cleave the IgG1 molecules.
Figure 3-9 – Distribution of soluble protein species in samples after light exposure determined by UV detection after SEC.
The content of the species is ranked on the left y-axis. The white bars represent the amount of fragments, the light grey bars represent the amount of dimers/trimers, the grey bars represent the amount of intermediate molecular weight aggregates (IMW) and the dark grey bars represent the high molecular weight aggregates (HMW). The right y-axis ranks the total recovery (black squares) and the monomer recovery (red dots). The error bars represent the standard deviation of at least two samples.
The appearance of aggregation and fragmentation potentially implicates changes in the conformation of the protein, detectable by spectroscopic methods analyzing the secondary and tertiary structure of a protein. The tertiary structure describes the steric constitution of the protein`s amino acid chain [Lottspeich, 2006]. Fluorescence spectroscopy and UV absorbance spectroscopy were utilized to investigate changes in the steric organization of the protein after light exposure. Intrinsic protein fluorescence was measured at 0.25 mg/mL protein
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concentration for the light exposed samples, since huge amounts of protein precipitated after 6 days of radiation and the concentration in the supernatant was too low for all intended analytical investigations. The intrinsic protein fluorescence is reduced with increasing duration of light exposure (see Figure 3-10a). After 1 day of light exposure, the fluorescence intensity slightly dropped. Further irradiation led to strong attenuation of the emission intensities.
However, no significant wavelength shift of the maximum emission peak around 337 nm was detected at any time point. The reduced intensities imply a quenching of Trp residues within the samples due to the exposure to more polar conditions, i.e. unfolding occurred.
Figure 3-10a – Intrinsic protein fluorescence of human IgG1 at 0.25 mg/mL after light exposure and excitation at 295 nm.
The black solid line represents the unstressed huAb sample, the grey solid line represents the shielded control sample and the dashed lines represent the light exposed sample after 1 d (red), 4 d (blue), 6 d (green), and 7 d (yellow). Each line represents the mean of at least two spectra recordings.
Differences were additionally detected in the section of the spectra displaying wavelengths higher than 380 nm. The emitting fluorescence intensities of samples radiated 4 days or longer increased in this section. This new emerging peak indicates the enhanced formation of dityrosine due to oxidation caused by light irradiation [Malencik et al., 1991;
Malencik et al., 1996]. Even more, a pronounced blue shift in wavelength of the dityrosine peak was monitored, proving that considerable changes in the polarity of the environment appeared additionally. The evident formation of novel species like dityrosine accompanied by the shift of peak position, emphasize that oxidation processes were induced by light exposure. The intrinsic protein fluorescence spectra also reveal that substantial structural changes started to appear at day 4 of light exposure, proceeding on days 6 and 7. The positions of dityrosine peak determined within the samples after several days light exposure are listed in Table 3-5.
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Table 3-5 – Results of secondary peak position from intrinsic protein fluorescence emission spectra of 0.25 mg/mL huAb after light exposure.
Light exposure Unstressed 4 d 6 d 7 d Control
Peak position 411.5 nm 423.9 nm 419.1 nm 411.1 nm 410.5 nm
The second derivative UV absorbance spectra were additionally monitored to investigate the tertiary structure after light exposure. The resulting spectra showed no shift in the wavelengths of any minimum or maximum peak between 280 and 300 nm (data not shown).
Differences were measured in the absorbance units of these minima and maxima. The a/b ratios calculated for the mean spectra are listed in Table 3-6. They show a significant trend to higher values with increasing duration of light exposure, suggesting that tyrosine residues are more and more exposed to the solvent [Ragone et al., 1984]. This increased exposure to the solvent indicates the unfolding of the human antibody.
Table 3-6 – Results of a/b ratio determination from UV 2nd derivative spectroscopy of huAb after light exposure.
Light exposure Unstressed 1 d 4 d 6 d 7 d Control
a/b ratio 1.4937 1.5771 1.8611 1.9301 2.0499 1.5152
Alterations in the conformational structure of the human antibody after light exposure were also detected by extrinsic fluorescence spectroscopy using 20 µM ANS and a protein concentration of 0.5 mg/mL. The emission spectra recorded of the supernatant of centrifuged samples are shown in Figure 3-10b.
Figure 3-10b – ANS fluorescence emission spectra of light exposed huAb samples at 0.25 mg/mL after excitation at 350 nm.
The solid black line represents the unstressed huAb sample, and the solid grey line represents the control samples, shielded from light. The light exposed samples are shown in dashed lines: in red (1 d), in blue (4 d), in green (6 d) and in yellow (7 d).
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Already after one day of light exposure the ANS fluorescence intensity is significantly enhanced. Between day 1 and day 4 the major enhancement of intensity was detected, whereas the further increases induced by the samples that were radiated even longer are relatively small. The positions of the ANS emission peaks are listed in Table 3-7. Ignoring the maximum intensity determined for the unstressed sample, a blue shift of the ANS-fluorescence maxima was detected. Between day 1 and day 7 a shift of nearly 10 nm was detected. The maximum detected in the unstressed sample might be imprecise due to the broad peak shape.
The strong increase in intensity as well as the detected shift of the maximum peak to lower wavelengths indicates pronounced alterations in the protein structure and unfolding processes induced by light irradiation. Within the control samples shielded from light a marginal increase of ANS-fluorescence intensity was detected. The determination of the peak maximum of this sample was imprecise as well, due to the peak shape, and thus it is not mentioned in Table 3-7.
Table 3-7 – Results of peak position from extrinsic ANS fluorescence emission spectra of 0.5 mg/mL huAb after light exposure and excitation at 350 nm.
Light exposure Unstressed 1 d 4 d 6 d 7 d Control
Peak position 475.5 nm 486.6 nm 478.9 nm 478.5 nm 477.0 nm 481.9 nm
Potential changes in the secondary structure were analyzed by FTIR spectroscopy of the amide I band. The second derivate spectra of the normalized zero-order spectra are shown in Figure 3-11. The dominating valley at 1638 cm-1 representing intramolecular β-sheets decreases with increasing light exposure. The decrease is first detectable on day 4 and accompanied by an increase in the range of 1650 – 1660 cm-1, an area that represents α-helical structures [Mahler et al., 2010a]. Even more, a second valley starts to form around 1634 cm-1, and the small dent at 1614 cm-1 is deepened and shifted to lower wavenumbers. These alterations indicate the formation of intermolecular β-sheet structures [Van De Weert et al., 2005]. The section of high wavenumbers (between 1660 and 1690 cm-1) within the second derivative spectra heavily changes due to light exposure, but also in the control sample shielded from light radiation. These regions are assigned to various secondary structures like loops, β-turns, and unordered structures [Mahler et al., 2010a; Van De Weert et al., 2005]. The control samples show also the beginning formation of an additional band between 1620 and 1630 cm-1, which is attributed to the formation of intermolecular β-sheets.
Light exposure has a strong influence on the secondary structure of a protein leading to numerous alterations. In summary, the predominant β-sheet structures of the investigated monoclonal antibody are disturbed and accompanied by an increase in α-helical structures
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Figure 3-11 – Second derivative ATR-FTIR spectra of light exposed huAb samples at 0.5 mg/mL.
The spectrum of the native huAb is plotted in the black solid line. The grey solid line represents the control samples after 7 days of incubation. The light exposed samples are plotted in dashed lines. Red = 1 d of light exposure, blue = 4 d of light exposure, green = 6 d of light exposure, and yellow = 7 d of light exposure.